Thirteen novel canine dog leukocyte antigen-88 alleles identified by sequence-based typing.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Canine DLA-79 gene: an improved typing method, identification of new alleles and its role in graft rejection and graft-versus-host disease.

Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (∼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.

CD34 cell dose in granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell grafts affects engraftment kinetics and development of extensive chronic graft-versus-host disease after human leukocyte antigen-identical sibling transplantation.

A retrospective analysis of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cell (G-PBMC) products harvested from healthy donors indicates significant variability in both the absolute number and relative proportion of CD34, CD3, and CD14 cells obtained. This report examined whether variations in the cellular composition of G-PBMC products correlated with clinical outcomes after myeloablative allogeneic transplantation. The numbers of CD34, CD3, and CD14 cells infused into 181 human leukocyte antigen (HLA)-identical sibling recipients were analyzed with respect to tempo of engraftment, acute graft-versus-host-disease (GVHD), clinical extensive chronic GVHD, overall survival, and disease relapse. Neither acute GVHD, overall survival, nor disease relapse was statistically significantly associated with CD34, CD3, or CD14 cell doses or the CD14 to CD3 ratio. CD3 and CD14 cell doses and CD14 to CD3 ratios did not correlate with the tempo of neutrophil and platelet engraftment. However, increasing CD34 cell numbers were significantly associated with accelerated neutrophil (P =.03) and platelet (P =.01) engraftment. Higher doses of CD34 cells (> 8.0 x 10(6)/kg) were also associated with a significantly increased hazard of clinical extensive chronic GVHD (HR = 2.3, 95% confidence interval [CI] 1.4-3.7, P =.001), but neither CD3 nor CD14 doses were statistically significantly associated with chronic GVHD. It was concluded that CD34 cell dose in G-PBMC grafts appears to affect both the engraftment kinetics and the development of clinical extensive chronic GVHD in HLA-identical sibling recipients but without a demonstrable impact on survival, relapse, and acute GVHD. Given the morbidity associated with extensive chronic GVHD, efforts to further accelerate engraftment in HLA-matched sibling transplants by increasing CD34 cell number in G-PBMC products may be counterproductive.

Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis.

A prospective randomized trial has shown that there is a survival advantage for allogeneic transplant recipients who received granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells (GPBMC) versus those who received bone marrow (BM) as a source of stem cells. The biological basis for this advantage is not clear and may be attributable to qualitative as well as quantitative differences in the CD34 cells, T cells, and/or the monocytes transplanted. To begin to address this issue, gene expression patterns in CD34 cells isolated from these 2 stem cell sources were compared to identify functional pathways that may distinguish these 2 populations. CD34 cells were isolated to purity from the BM and peripheral blood stem cells of multiple healthy donors. (The complete data set will be available at http://parma.fhcrc.org/lgraf upon publication.) Two separate RNA preparations from pooled samples from both sources were analyzed by Affymetrix Oligonucleotide Array chips for expression of over 6400 human genes. Comparative analyses among the samples showed that a small set of 28 sequences increased and 38 sequences decreased in expression more than 3-fold in both of the GPBMC samples compared to those in BM samples. More highly expressed genes include several for nuclear proteins and transcriptional factors. Functional categorization of the genes decreased in expression indicated sequences influential in cell cycle progression, in agreement with the recognized quiescence of circulating CD34 cells. Multiple transcriptional regulators and chemokines were also found to be decreased. These data emphasize that in addition to increased numbers of CD34 cells, G-CSF mobilization also results in significant qualitative changes. Whether they impact engraftment remains to be determined.

Hematopoietic responses to stress conditions in young dogs compared with elderly dogs.

Clinical observations show that older patients do not tolerate high-dose chemoradiotherapy as well as younger patients. It is unclear whether this is due to age-related differences in their responses to hematopoietic injury or to differential toxicities to other organs. In the present study, 6 young (0.5 years) and 6 elderly (8 years) dogs were challenged with 7 repeated nonlethal doses of 50 or 100 cGy total body irradiation (TBI) each (total 550 cGy), and 21 days of recombinant canine granulocyte-colony stimulating factor (rcG-CSF) after the last TBI dose. Recoveries of absolute neutrophil, platelet, and lymphocyte counts after each TBI dose, responses to rcG-CSF treatment, and telomere lengths in neutrophils were compared before and after the study. No differences were found in recoveries of neutrophils, platelets, or in responses to rcG-CSF among young and old dogs. In contrast, recoveries were suggestively worse in younger dogs. After rcG-CSF, platelet recoveries were poor in both groups compared with previous platelet recoveries (P <.01). Consequently, 2 old and 3 young dogs were euthanized because of persistent thrombocytopenia and bleeding. At the study's completion, marrow cellularities and peripheral blood counts of the remaining young and elderly dogs were equivalent. The telomere lengths in both groups were significantly reduced after the study versus beforehand (P =.03), but the median attritions of telomeres were not different. It was concluded that aging does not appear to affect hematopoietic cell recoveries after repeated low-dose TBI, suggesting that poor tolerance of radiochemotherapy regimens in older patients may be due to nonhematopoietic organ toxicities rather than age-related changes in hematopoietic stem cells reserves.

Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects.

Toxicities have limited the use of allogeneic hematopoietic cell transplantation (HCT) to younger, medically fit patients. In a canine HCT model, a combination of postgrafting mycophenolate mofetil (MMF) and cyclosporine (CSP) allowed stable allogeneic engraftment after minimally toxic conditioning with low-dose (200 cGy) total-body irradiation (TBI). These findings, together with the known antitumor effects of donor leukocyte infusions (DLIs), led to the design of this trial. Forty-five patients (median age 56 years) with hematologic malignancies, HLA-identical sibling donors, and relative contraindications to conventional HCT were treated. Immunosuppression involved TBI of 200 cGy before and CSP/MMF after HCT. DLIs were given after HCT for persistent malignancy, mixed chimerism, or both. Regimen toxicities and myelosuppression were mild, allowing 53% of eligible patients to have entirely outpatient transplantations. Nonfatal graft rejection occurred in 20% of patients. Grades II to III acute graft-versus-host disease (GVHD) occurred in 47% of patients with sustained engraftment. With median follow-up of 417 days, survival was 66.7%, nonrelapse mortality 6.7%, and relapse mortality 26.7%. Fifty-three percent of patients with sustained engraftment were in complete remission, including 8 with molecular remissions. This novel allografting approach, based on the use of postgrafting immunosuppression to control graft rejection and GVHD, has dramatically reduced the acute toxicities of allografting. HCT with the induction of potent graft-versus-tumor effects can be performed in previously ineligible patients, largely in an outpatient setting. Future protocol modifications should reduce rejection and GVHD, thereby facilitating studies of allogeneic immunotherapy for a variety of malignancies. (Blood. 2001;97:3390-3400)

G-CSF-mobilized peripheral blood mononuclear cells added to marrow facilitates engraftment in nonmyeloablated canine recipients: CD3 cells are required.

Stable mixed donor/host hematopoietic chimerism can be uniformly established in dogs conditioned with 200 cGy TBI before dog leukocyte antigen (DLA)-identical marrow transplantation and immunosuppressed with a short course of mycophenolate mofetil (MMF) and cyclosporine (CSP) after the transplantation. A further decrease in the TBI dose to 100 cGy or the elimination of MMF in this model results in graft rejection. Here we asked whetherthe addition of G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) to marrow grafts would enhance donor engraftment in dogs conditioned with 100 cGy TBI and given postgrafting immunosuppression with CSP alone. Using this model, 7 of 9 dogs given only marrow cells rejected their grafts within 8 to 17 weeks after transplantation. In contrast, the addition of unmodified G-PBMC to marrow grafts resulted in stable mixed donor/host chimerism in 5 of 8 dogs studied (P = .06). However, addition of the CD3-depleted fraction of G-PBMC, which contained both CD34 cells and CD14 cells, resulted in engraftment in only 1 of 7 recipients. We conclude that adding G-PBMC to marrow grafts replaced the requirement of MMF and 100 cGy of TBI, and that CD3 cells were required to facilitate engraftment of marrow cells in DLA-identical recipients, whereas the additional CD34 cells present in G-PBMC were not sufficient for this effect.

Characterization and functional analysis of laminin isoforms in human bone marrow.

Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed alpha, beta, and gamma chains. Combinations of 11 characterized laminin subunits (alpha 1-alpha 5, beta 1-beta 3, and gamma 1-gamma 3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase-polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34(+) cell lines. Antibodies against the beta 1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells. (Blood. 2000;96:4194-4203)